The methods and system of the present invention employ optical, or spectroscopic, detection techniques for assessing the health physiological condition, and viability of biological materials such as tissues, cells, and subcellular components, and may be used in both in vitro and in vivo systems. One important application of the methods and apparatus of the present invention is high throughput screening of candidate agents and conditions to evaluate their suitability as diagnostic or therapeutic agents.
Biology has undergone a change so fundamental that it has been compared to the industrial revolution of the 19th century and the advances in quantum physics in the 20th, century. The complete sequencing of the human genome and of the genomes of many microbes and plants has given rise to genomics, the discipline defined as the study of the structure and function of large number of genes undertaken in a simultaneous fashion. While the value of genomics as a basic tool for biological research has been clearly demonstrated, the impact on drug discovery remains unrealized. It is now clear that knowledge of gene sequences does not imply an understanding of their function. For example, inferred function based on the study of homologs from model organisms allows only the assignment of function to approximately 10% of the genes of mouse and humans. Clearly, the understanding of the function of genes at all organizational levels of biology will be the primary challenge of the post-genomics era.
The breadth and scale of the research efforts in genomics and related disciplines has resulted in the generation of large quantities of data that are difficult to examine or understand. Evolving relational databases will include not just primary biological data, but also predictions of protein structure, dynamic models of complex physiological processes, and the statistical treatment of data. The evolution of bioinformatics has occurred in parallel with the creation of increasingly sophisticated tools for compiling and analyzing chemical data. Theoretical and experimental chemical data is being incorporated into advanced chemical databases that are being relationally connected to genomic databases to give rise to the field of chemical genomics. Finally, engineering and the physical sciences play a crucial role in the post-genomics era. Advances in analytical chemistry, analytical biochemistry, image analysis, robotics and process automation have enabled the task of developing advanced biological databases.
In spite of the availability of numerous targets for drug discovery, the overall success rate of the process remains abysmally low. At present, it is fair to say that the question How does one apply information about gene and gene products to the discovery of new drugs remains largely unanswered. There are three main reasons for low success rates in the conversion of vast amounts of genomics information to viable products: (1) lack of clear criteria for target validation; (2) hits to leads decisions based on potency and selectivity against molecular targets, with limited physiological information; and (3) nonviable leads due to poor adsorption, undesirable metabolism, toxicity, or unacceptable side effects.
Drug development programs rely on in vitro screening assays and subsequent testing in appropriate animal models to evaluate drug candidates prior to conducting clinical trials using human subjects. Screening methods currently used are generally difficult to scale up to provide the high throughput screening necessary to test the numerous candidate compounds generated by traditional and computational means. Moreover, studies involving cell culture systems and animal model responses frequently don""t accurately predict the responses and side effects observed during human clinical trials.
Conventional methods for assessing the effects of various agents or physiological activities on biological materials, in both in vitro and in vivo systems, generally are not highly sensitive or informative. For example, assessment of the effect of a physiological agent, such as a drug, on a population of cells or tissue grown in culture, conventionally provides information relating to the effect of the agent on the cell or tissue population only at specified points in time. Additionally, current assessment techniques generally provide information relating to a single or a small number of parameters. Candidate agents are systematically tested for cytotoxicity, which may be determined as a function of concentration. A population of cells is treated and, at one or several time points following treatment, cell survival is measured. Cytotoxicity assays generally do not provide any information relating to the cause(s) or time course of cell death.
Similarly, agents are frequently evaluated based on their physiological effects, for example, on a particular metabolic function or metabolite. An agent is administered to a population of cells or a tissue sample, and the metabolic function or metabolite of interest is assayed to assess the effect of the agent. This type of assay provides useful information, but it does not provide information relating to the mechanism of action, the effect on other metabolites or metabolic functions, the time course of the physiological effect, general cell or tissue health, or the like.
Optical techniques have been developed and used for several applications. Light scattering has been used in the past to provide measurements of osmotic water permeability in suspensions of osmotically responsive vesicles and small cells. A. S. Verkman, xe2x80x9cOptical Methods to Measure Membrane Transport Processes,xe2x80x9d J. Membrane Biol. 148:99-110, 1995. Another study reported a method for the optical measurement of osmotic water transport in cultured cells. M. Echevarria, A. S. Verkman, xe2x80x9cOptical Measurement of Osmotic Water Transport in Cultured Cells: Role of Glucose Transporters,xe2x80x9d J. Gen. Physiol. 99:573-589, 1992.
Optical techniques for observing nerve activity and neuronal tissue are well-established. Hill and Keynes observed that the nerve from the walking leg of the shore crab normally has a whitish opacity caused by light scattering, and that opacity changes evoked by electrical stimulation of that nerve were measurable. Hill, D. K. and Keynes, R. D., xe2x80x9cOpacity Changes in Stimulated Nerve,xe2x80x9d J. Physiol. 108:278-281, 1949. Since the publication of those results, experiments designed to learn more about the physiological mechanisms underlying the correlation between optical and electrical properties of neuronal tissue and to develop improved techniques for detecting and recording activity-evoked optical changes have been ongoing.
Intrinsic changes in optical properties of cortical tissue have been assessed by reflection measurements of tissue in response to electrical or metabolic activity. Grinvald, A., et al., xe2x80x9cFunctional Architecture of Cortex Revealed by Optical Imaging of Intrinsic Signals,xe2x80x9d Nature 324:361-364, 1986; Grinvald, et al., xe2x80x9cOptical Imaging of Neuronal Activity, Physiological Reviews, Vol. 68, No. 4, October 1988. Grinvald and his colleagues reported that some slow signals from hippocampal slices could be imaged using a CCD camera without signal averaging.
A CCD camera was used to detect intrinsic signals in a monkey model. Ts""o, D. Y., et al., xe2x80x9cFunctional Organization of Primate Visual Cortex Revealed by High Resolution Optical Imaging,xe2x80x9d Science 249:417-420, 1990. The technique employed by Ts""o et al. would not be practical for human clinical use, since imaging of intrinsic signals was achieved by implanting a stainless steel optical chamber in the skull of a monkey and contacting the cortical tissue with an optical oil. Furthermore, in order to achieve sufficient signal to noise ratios, Ts""o, et al., had to average images over periods of time greater than 30 minutes per image.
The mechanisms responsible for intrinsic signals are not well understood. Possible sources of intrinsic signals include dilation of small blood vessels, changes in blood flow, volume and oxygenation, neuronal activity-dependent release of potassium, and swelling of neurons and/or glial cells caused, for example, by ion fluxes or osmotic activity. Light having a wavelength in the range of 300 to 3000 nm may also be reflected differently between active and quiescent tissue due to increased blood flow into regions of higher neuronal activity. Yet another factor that may contribute to intrinsic signals is a change in the ratio of oxyhemoglobin and deoxyhemoglobin in blood.
U.S. Pat. No. 5,215,095 discloses methods and apparatus for real time imaging of functional activity in cortical areas of a mammalian brain using intrinsic signals. A cortical area is illuminated, light reflected from the cortical area is detected, and digitized images of detected light are acquired and analyzed by subtractively combining at least two image frames to provide a difference image. U.S. Pat. Nos. 6,196,226 and 6,233,480 disclose similar optical methods and apparatus for optical detection of neuronal tissue and activity.
U.S. Pat Nos. 5,438,989 discloses a method for imaging margins, grade and dimensions of solid tumor tissue by illuminating the area of interest with high intensity electromagnetic radiation containing a wavelength absorbed by a contrast agent, obtaining a background video image of the area of interest, administering a contrast agent, and obtaining subsequent video images that, when compared with the background image, identify the solid tumor tissue as an area of changed absorption. U.S. Pat. Nos. 5,699,798 and 6,241,672 disclose methods and apparatus for optically distinguishing between tumor and non-tumor tissue, and imaging margins and dimensions of tumors during surgical or diagnostic procedures.
U.S. Pat. No. 5,465,718 discloses a method for imaging tumor tissue adjacent to nerve tissue to aid in selective resection of tumor tissue using stimulation of a nerve with an appropriate paradigm activate the nerve, permitting imaging of the active nerve. The ""718 patent also discloses methods for imaging of cortical functional areas and dysfunctional areas, methods for visualizing intrinsic signals, and methods for enhancing the sensitivity and contrast of images. U.S. Pat. Nos. 5,845,639 and 6,161,031 disclose optical imaging methods and apparatus for detecting differences in various blood parameters, such as blood flow rates and flow changes, as well as cortical areas of neuronal inhibition.
U.S. Pat. Nos. 5,902,732 and 5,976,825, which are related to this application, disclose methods for screening drug candidate compounds for anti-epileptic activity using glial cells in culture by osomotically shocking glial cells, introducing a drug candidate, and assessing whether the drug candidate is capable of abating changes in glial cell swelling. These patents also disclose a method for screening drug candidate compounds for activity to prevent or treat symptoms of Alzheimer""s disease, or to prevent CNS damage resulting from ischemia, by adding a sensitization agent capable of inducing apoptosis and an osmotic stressing agent to CNS cells, adding the drug candidate, and assessing whether the drug candidate is capable of abating cell swelling. U.S. Pat. Nos. 6,096,510 and 6,319,682, which are also related to this application, disclose additional methods and systems for assessing biological materials using optical detection techniques. A method for determining the viability and health of living cells inside polymeric tissue implants is also disclosed, involving measuring dimensions of living cells inside the polymeric matrix, osmotically shocking the cells, and then assessing changes in cell swelling. Assessment of cell swelling activity is achieved by measuring intrinsic optical signals using an optical imaging screening apparatus.
The methods and systems of the present invention are directed to problems that present fundamental limitations in our ability to make the step from the level of genomics and proteonomics to the design of novel therapeutics. The solution to these problems necessarily requires non-destructive methods for extracting quantitative biological responses in living, physiologically meaningful systems and environments. The physiomics approach, described herein, has several requirements that are entirely unique, and set it apart from technologies and methods conventionally applied to molecular biology. This is because the physiological responses in cells and tissues are both spatially and temporally dynamic, and hence require technologies that can monitor changes in living systems that are occurring in distinct spatial locations with high temporal resolution.
Cells from nearly every organ and tissue, of both plant and animal origin, can be dissociated into single cells, grown and propagated using cell culture techniques. Pathological cells from diseased or dysfunctional tissue can also be isolated and maintained in tissue culture. Various types of tissues, including normal, dysfunctional and malignant tissues, may be maintained under culture conditions for prolonged periods of time and assessed according to methods of the present invention. Short-term experiments may also be conducted on living acute tissue samples that are prepared and maintained under physiological conditions. Many multicellular systems and tissues may also be maintained as functioning systems in cell culture. Healthy, pathogenic and dysfunctional cells and tissue may also be tested and observed in situ in living animal models, including humans, using methods and systems of the present invention.
All cells undergo physiological processes that contribute to and determine their geometrical structure and optical properties. These physiological processes include metabolic processes, volume-regulatory responses, gene expression, endocytosis, pinocytosis, ion homeostasis, immune responses, neurological activity and inhibition, responses to mechanical trauma, chemical and environmental insult, and the like. Various events, including disease states, dysfunction, inflammation, exposure to pathogens, pollutants, radiation, chemotherapy, infectious or other agents, aging, apoptosis, necrosis, oncogenesis, genetic modification, and the like, affect one or more of these physiological processes, producing measurable and predictable changes in the characteristic geometrical structure and/or optical properties of individual cells and/or cell populations.
The methods and systems of the present invention employ optical, or spectroscopic, detection techniques to assess the physiological state of biological materials including cells, tissues, organs, subcellular components and portions of intact organisms. The biological materials may be of human, animal, bacterial, viral or plant origin, or they may be derived from any such materials. Static and dynamic changes in the geometrical structure and/or optical properties of the biological materials in response to the administration of a physiological challenge or a test agent are indicative and predictive of changes in the physiological state or health of the biological material. The physiological responses of cells and tissues to the administration of a physiological challenge or a test agent are both spatially and temporally dynamic, and require monitoring of the changes in living systems with high spatial and temporal resolution. The methods and systems of the present invention provide the technology for monitoring changes in living systems with high spatial and temporal resolution.
Numerous physiological states and conditions may be assessed in cell sample populations, and in situ, in living biological systems, using the optical techniques of the present invention. Cell viability and identification of non-viable and viable cells and tissues may be assessed using the optical techniques described, for example, in U.S. Pat. No. 6,096,510. Tissue hemodynamics, including blood volume and blood oxygenation, may be assessed using the optical techniques described, for example, in U.S. Pat. Nos. 5,845,639 and 6,161,031. Tumor tissue may be identified and characterized using the optical techniques described, for example, in U.S. Pat. Nos. 5,699,798 and 6,241,672. CNS and peripheral nerve activity and inhibition, including neuronal excitability and synchronization, may be assessed using the optical techniques described, for example, in U.S. Pat. Nos. 6,196,226 and 6,233,480. Apoptotic and necrotic cell death processes may be observed and distinguished using the methods and systems described herein. Cell volume regulation and changes are observed as changes in intrinsic optical properties of cell sample populations. Changes in the intrinsic optical properties of cell sample populations, measured as changes in light scattering and absorption, are also indicative and predictive of the physiological state and condition of cell sample populations.
Assessment of intracellular calcium, intracellular pH, intracellular chloride, ATP levels, ATP/ADP ratios, cyclic AMP, oxidative activity, intracellular reactive oxygen species, metabolic state of mitodhondria, cystolic redox potential, nitric oxide, cell motility, mitochondrial transmembrane potential, mitochondrial swelling, lysosome activity, intracellular zinc, intracellular magnesium, intracellular sodium, intracellular potassium, cell membrane potential, cell proliferation and many other cellular, intracellular and biological system conditions, are also observed as changes in intrinsic and/or extrinsic optical properties of cell sample populations. The methods and systems of the present invention thus provide the capability to screen numerous cell populations, tissue types and biological systems and acquire data relating to a wide variety of cellular, intracellular and biological systems conditions.
Another aspect of methods and systems of the present invention involves the development and use of databases cataloging the complex responses of various biological materials including cells, tissues, organs, subcellular components, portions of intact organisms, and the like, to various physiological challenges or test agents. Changes in the geometrical and/or optical properties of living systems induced (or not) by admininstration of a physiological challenge or a test agent are detected with a high degree of spatial and temporal resolution, stored, compared to changes induced in different biological systems and/or by administration of different physiological challenges and/or test agents, and patterns are identified. The changes induced by different physiological challenges and/or test agents on specific biological sample materials have unique profiles that are predictive of various types of responses and are useful for the development and screening of candidate therapeutic and diagnostic compositions.
The database may archive, for example, the spatially and temporally resolved response patterns of cell, tissue and whole animal sample populations to existing, approved therapeutic agents. The spatially and temporally resolved response patterns of candidate agents and combinations may then be compared to and screened against the response patterns of approved and presumably safe and efficacious known agents to predict the response of the candidate agent and/or combination in a biological system. Comparison of screening data against response patterns stored in the database provides valuable assessment of target validation, lead selection and optimization, and detection of side effects.
Two different classes of dynamic phenomena are observed in viable biological materials using optical detection techniques: (1) geometrical changes in the diameter, volume, conformation, intracellular space of individual cells or extracellular space surrounding individual cells; and (2) changes in one or more optical properties of individual cells, intracellular structures or of cell populations. The changes in optical properties may be intrinsic optical changes, such as light scattering, reflection, absorption, refraction, diffraction, birefringence, refractive index, Kerr effect, and the like, that are indicative of various physiological conditions. Alternatively, changes in optical properties may be induced by administration of a test agent such as a dye or another type of contrast agent, such changes in optical properties including changes in absorption, scattering, birefringence, fluorescence, and phosphorescence. Both classes of phenomena may be observed statically or dynamically, with or without the aid of a contrast enhancing agent. Geometrical changes may be assessed directly by measuring (or approximating) the geometrical properties of individual cells, or indirectly by observing changes in the optical properties of cells. Changes in optical properties of individual cells or cell populations may be assessed directly using systems of the present invention.
Observation and interpretation of geometrical and/or optical properties of individual cells and cell populations is achieved in both in vitro and in vivo biologically viable systems without altering characteristics of the sample by applying physiologically invasive materials, such as fixatives, and without using ionizing radiation, microelectrode penetration, or other techniques that are damaging or destructive to the cell population. Physiologically non-invasive contrast enhancing agents, such as vital dyes, markers, probes, and the like, may be used in desired applications to enhance the sensitivity of optical detection techniques. In applications employing contrast enhancing agents, the optical detection techniques are used to assess extrinsic optical properties of the biological materials.
Detection and analysis of the geometrical and/or intrinsic optical properties of individual cells or sample cell populations using techniques that provide a high level of spatial and temporal resolution provides information permitting the classification of the physiological state of individual cells or sample cell populations. Based on analysis of the geometrical and/or optical properties of a sample cell population, the sample may be classified as viable or non-viable, apoptotic, necrotic, proliferating, in a state of activity, inhibition, synchronization, or the like, or in any of a variety of physiological states, all of which produce distinct geometrical and/or optical profiles. The use of probes and labeled markers and assaying of extrinsic optical properties of sample populations to assess various physiological conditions at resolved locations in time and in space provides yet further information relating to cellular and biological systems responses to various physiological challenges and test agents.
An important application of the methods and systems of the present invention involves screening cell populations to assess the effect(s) of exposure to various types of test agents or test conditions, including candidate compounds and combinations, drugs, hormones and other biological agents, toxins, infectious agents, physiological stimuli, radiation, chemotherapy, and the like. Safety and cytotoxicity testing is conducted by exposing a sample population to a test agent or test condition and assessing the physiological state of the sample population using optical techniques at one or more time points following administration of the test agent or test condition. The effect of various test agents and conditions may be evaluated on numerous types of normal and pathological sample populations. Such testing may be conducted on various sample populations to determine how a test agent or condition affects a desired target sample population, as well as to predict whether a test agent or condition produces physiological side effects on sample populations that are not the target of the test agent or condition.
Thus, for example, a candidate for treatment of central nervous system (CNS) conditions may be screened using various types of CNS cell and tissue populations to determine safety and efficacy in the CNS, as well as other types of cell populations, such as renal or hepatic cell populations, to determine safety and efficacy in other portions of the biological system. Similarly, a candidate for treating a cancer may be screened using various types of cancer and normal cell populations to assess the specificity and toxicity of the candidate agent or combination on various cell populations. Additionally, the effect of genetic modifications on the numerous physiological states and conditions described herein may be assessed with respect to various cell and tissue sample populations and compared to unaltered, wild-type sample populations, or to differently genetically altered sample populations.
In addition to assessing the safety and efficacy of a candidate composition or combination, numerous physiological responses of the sample population to the agent, including those described above, may be determined and assessed. Thus, various intracellular responses, such as changes in intracellular properties, such as pH, reactive oxygen species, calcium, chloride, ATP, zinc, magnesium, sodium, potassium, and the like, may be assessed, in addition to changes in other cell-based properties, such as cell membrane potentials and the like, in addition to various cellular and environmental properties, such as changes in intracellular and extracellular volume, light scattering, and the like, in addition to gross changes in the cell sample population, such as changes in viability, apoptotic and necrotic activity. All of these properties and responses may be assessed using the optical methods and systems of the present invention. Analysis of these properties is predictive of the behavior and effect of a candidate composition or combination in a biological system, such as an animal. In yet another aspect, profiles determined for various candidate agents in various types of sample populations may be compared to profiles for various types of sample populations established for other treatment agents having known biological effects. Comparison and analysis of such profiles and patterns within profiles provides accurate prediction of the biological effects of candidate agents within a biological system.
According to one embodiment, a disease state or compromised condition is simulated in biological materials prior to administration of a test agent or test condition to assess the suitability of the test agent or condition for treating the disease state or compromised condition. Exposure of sample populations to a physiological challenge, such as a change in extracellular osmolarity or ion concentration, altered oxygen or nutrient or metabolite conditions, drugs or diagnostic or therapeutic agents, a disturbance in ion homeostasis, electrical stimulation, inflammation, infection with various agents, radiation, and the like, simulates a disease state at a cellular or tissue level. Subsequent exposure of the sample populations a test agent or condition and detection and analysis of changes in geometrical and/or optical properties of the sample populations provides information relating to the physiological state of the sample populations produced by the test agent or condition. Screening techniques may be adapted for use with various types of cell sample populations maintained in vitro under appropriate cell culture conditions to provide a high throughput, automated screening system. Alternatively, screening techniques may be adapted to examine cell and tissue populations using various animal models to assess the effect of a physiological challenge and/or administration of a test agent on various cell populations in animal models in situ. Screening techniques of the present invention may also be implemented to examine cell and tissue populations, using animal and plant models, to assess the effect(s) of genetic modifications of such animal and plant models in situ.
Changes in geometrical and/or optical properties of individual cells or cell sample populations may be compared to empirically determined standards for specific cell types, cell densities and various physiological states, or appropriate controls may be run in tandem with the test samples to provide direct comparative data. Data corresponding to spatial and temporal changes in geometrical and/or optical properties of sample populations is collected and, preferably, stored to provide data relating to the time course and spatial distribution over the time course of the effect of a test agent or condition on sample populations. Strategies for designing screening protocols, including appropriate controls, multiple samples for screening various dosages, activities, and the like, are well known in the art and may be adapted for use with the methods and systems of the present invention.